Seed maturation proteins, or late embryogenesis abundant (LEA) proteins, are produced in abundance during the late drying phase of seed development. Most LEA proteins accumulate in seeds or vegetative tissues that were exposed to exogenous abscisic acid or that undergo abiotic stress caused by salinity or dehydration. Some LEA proteins confer salt and dehydration tolerance in transgenic plants, probably by hydrating macromolecules, sequestering ions and renaturing unfolded proteins.
Using suppression subtractive techniques, nine genes were found to be more highly expressed under abiotic stress conditions than under normal conditions. One of them (GmDNJ1) shared 99% nucleotide sequence homology to Glycine max seed maturation protein PM37 (GmPM37) deposited in GenBank as AF 169022, which is a DnaJ homolog. As shown in FIG. 11a, it contains the characteristic components of DnaJ including the conserved N-terminal J-domain, a glycine/phenylalanine rich domain, a domain that includes a (CXXCXGXG)4 (SEQ ID NO:1) zinc finger type motif and an uncharacterized C-terminal domain. (Cyr, D. M., et al., J. Biol. Chem. (1994) 269:9798-9804, Hennessy, F., et al., Cell Stress & Chaperones (2000) 4:347-358). GmDNJ1 did not resemble any LEA proteins.
DnaJ-like proteins are believed to serve as chaperone or co-chaperone proteins principally by aiding the chaperone function of Hsp70s. Hsp70 is one of many heat shock proteins originally found to occur in Drosophila larvae in response to elevated temperatures. Heat shock proteins in general, while they may be produced either constitutively or under stress conditions, are believed, to play a chaperone role.
DnaJ-like proteins are defined by a conserved “J” region of approximately 73 amino acids (based on the originally disclosed E. coli protein, typically occurring toward the N-terminus of the protein (Hennessy, F., et al., supra). This domain is slightly shorter in the eucaryotic counterparts. As noted by Hennessy, et al., DnaJ-like proteins have been classified as: Type I which contain similarity to DnaJ over all domains, including the J-domain, the glycine-phenylalanine rich domain, and the (CXXCXGXG)4 (SEQ ID NO:1) motif; Type II which contain the J-domain and the glycine-phenylalanine rich region; and Type III which contain only the J-domain. These groups have been renamed A, B and C, respectively.
The J-domain is required to include the triplet histidine-proline-aspartic (HPD) and also contains a number of other highly conserved regions. Hennessy, et al., (supra) in FIG. 3 provides detailed comparison of the J-domains of a number of DnaJ-like proteins indicating greater consensus in these regions among proteins of Types I and II than of Type III.
Miernyk, J. A., Cell Stress & Chaperones (2001) 6:209-218 using analysis of the genome and EST profiles of Arabidopsis thaliana shows that the genome encodes 89 J-domain containing proteins which correspond to varying levels of EST's. Of these, only one appears to be highly expressed; it is a Type III DnaJ-like protein. No analysis of actual protein levels was performed.
DnaJ expression has been reported to be associated with salt and dehydration tolerance in plants. Zhu, et al, Cell (1993) 5:341-349 showed that the expression of a DnaJ homologue from the higher plant Atriplex nummularia (ANJ1) was induced in plant cell culture under salinity stress. Recently, Nguyuen, et al., Mol. Gen. Genomics (2004) 272:35-46 developed a marker for mapping of quantitative trait loci (QTL) regions for dehydration tolerance in rice, which was shown to be similar to Zea mays DnaJ-related protein (ZMDJ1), which was induced by heat stress (Baszczynski, et al., Maydica (1997) 42:189-201). Although these studies reported the induction of DnaJ homologue under salt and dehydration stresses, no characterization of the effect of ANJ1 and ZMDJ1 on salt or dehydration tolerance was reported.
A group of seed maturation proteins includes small heat shock proteins (sHsps), but does not include DnaJ or Hsp40 (Wise, BMC Bioinformatics (2003) 29:52-70). Only GmPM37 (GmDNJ1) has been reported as a DnaJ-like seed maturation protein. The deduced protein sequence showed that GmDNJ1 contains the conserved motifs of DnaJ, and has a predicted molecular weight similar to common DnaJ proteins (Hdj1: 38 kDa; Ydj1: 45 kDa; Hsp40: 41 kDa).
The present applicants are not aware of any reports describing the functional role of DnaJ proteins in conferring tolerance in plants to abiotic stress except heat shock.